Hematopoiesis is the developmental process in which progenitor cells differentiate into different blood cell lineages. Ets proteins are critical to this process. The Ets protein PU.1 is expressed in B cells, monocytes, granulocytes, megakaryocytes, mast cells and immature erythrocytes. Spi-B, which is highly related to PU.1 is predominantly expressed in B cells. PU.1 and Spi-B bind the same DNA elements, so they may activate similar target genes. PU.1-/- mutant mice die at embryonic day 16.5 lacking B cells , T cells, monocytes and granulocytes. Spi-B /- mice are viable but exhibit functional defects in B cells. Given the distinct phenotypes exhibited by mutant mice, the experiments described in this proposal are designed to identify overlapping and unique functions of these related proteins. Spi-B and Ets-1 (an Ets protein highly divergent from PU.1) will be Inserted into the PU.1 locus in ES cells. To determine if Spi-B and Ets-1 complement PU.1 deficiency, these proteins will be analyzed by in vitro differentiation to look at myeloid cells. Next an in vitro B cell culture system and RAG chimera mice will be used to look at B cell rescue by Spi-B. Finally, mice with Spi-B in the PU.1 locus will be generated to determine if the embryonic lethality is rescued and if blood cells are produced in appropriate numbers. The experiments outlined above will enlighten us to the functions PU.1 and Spi-B share and the functions unique to each protein.